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3 Detection and Quantitation of Tetrahydrocannabinol in Physiological Fluids

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M O N R O E E. W A L L , D O L O R E S R. B R I N E , J O A N T . B U R S E Y , and D A V I D R O S E N T H A L Chemistry and Life Sciences Division, Research Triangle Institute, P. O . Box 12194, Research Triangle Park, NC 27709

In recent years there has been a great increase i n i n t e r e s t i n the pharmacology, metabolism and biodisposi tion of the cannabinoids; f o r recent reviews c f . Mechou lam ( 1 ) , Paton and Crown (2), Wall (3) and Wall et al. (4). U n t i l r e c e n t l y , q u a n t i t a t i o n o f the various canna binoids in b l o o d , u r i n e , feces and other b i o l o g i c a l t i s s u e s could be c a r r i e d out only by the use of appro­ priately r a d i o - l a b e l e d analogs of the cannabinoids un­ der study (4, 5). Because of the widespread and in­ creasing opposition to the use of r a d i o - l a b e l e d isotopes in studies i n v o l v i n g man and because many of the studies c u r r e n t l y being conducted w i t h various cannabinoids in volve large scale experiments in which r a d i o - l a b e l e d cannabinoids are not used, the need f o r the development of n o n - r a d i o - l a b e l e d q u a n t i t a t i v e methodology f o r cer tain key cannabinoids has become i n c r e a s i n g l y apparent. In a d d i t i o n , r a d i o - l a b e l e d t h i n l a y e r chromatography techniques, w h i l e useful in initial s t u d i e s , lack suf ficient accuracy. When biological e x t r a c t s are s t u d i e d by r a d i o - l a b e l e d means, separation of Δ -THC from canna b i n o l and of 11-hydroxy-Δ -THC from other monohydroxy l a t e d analogs is poor. I f such i n t e r f e r i n g substances are present in considerable q u a n t i t y , one will obtain erroneously high values. This will i n c r e a s i n g l y be the 9

9

0-8412-0488-8/79/47-098-039$05.00/0 © 1979 American Chemical Society

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

40

CANNABINOID

ANALYSIS

IN

PHYSIOLOGICAL

FLUIDS

case when one i s a n a l y z i n g b i o l o g i c a l m a t e r i a l s ob­ t a i n e d from m a r i j u a n a smokers w h i c h c o n t a i n A -THC, c a n n a b i n o l , c a n n a b i d i o l , and 1 1 - h y d r o x y l a t e d a n a l o g s o f t h e s e compounds. Q u a n t i t a t i v e gas l i q u i d chromatography combined w i t h mass s p e c t r o m e t r y (GLC-MS) has been used w i t h ex­ c e l l e n t r e s u l t s f o r the q u a n t i t a t i v e a n a l y s i s o f drugs i n b i o l o g i c a l m a t e r i a l s , c o m b i n i n g as i t does the s e p ­ a r a t i v e powers o f GLC and the i n h e r e n t s e n s i t i v i t y o f MS d e t e c t i o n . P i o n e e r s t u d i e s by Hammar and H o l m s t e d t (6) i n t r o d u c e d the c o n c e p t o f mass fragmentography (now a l s o c a l l e d m u l t i p l e i o n d e t e c t i o n (MID) ) and g r e a t l y i n c r e a s e d the s e n s i t i v i t y o f MS methodology so t h a t i t c o u l d be a p p l i e d t o the nanogram and p i c o g r a m l e v e l s . The c o n c e p t has been a p p l i e d t o many drugs i n c l u d i n g a r e c e n t use by A g u r e l l , H o l m s t e d t and c o - w o r k e r s (7) i n the d e t e r m i n a t i o n o f A -THC i n b l o o d and plasma. The a p p l i c a t i o n o f GLC-MS t e c h n i q u e s t o the in_ v i t r o and i l l v i v o m e t a b o l i s m o f A -THC (8) l a i d the ground­ work f o r the q u a n t i t a t i v e a n a l y s i s o f o t h e r cannabinoid metabolites. The s i t e s o f m e t a b o l i c h y d r o x y l a t i o n f o r many c a n n a b i n o i d s a r e shown i n F i g u r e 1.

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9

9

9

L

Hydroxylation in

vivo

in

other

quently

singly

at

11

or

position

in

positions.

observed

combination Oxidation

to

In

with

vitro

and

hydroxylation

carbonyl

also

fre­

observed. 9



Hydroxylation

at

8

series*,



both

8a

or

8,11-dihydroxy 1

7α,



or

position

metabolites

7,11-Dihydroxy ,

*

Figure 1.

found

found as

only

minor

only

Δ

-THC

metabolites*

frequently

found

in

in

observed. g Δ -series.

,

H y d r o x y l a t i o n at 1 ,2*,3 ,4' o c c a s i o n a l l y found, a l s o as Ι',ΙΙ; 2 ' , 1 1 - e t c . d i h y d r o x y m e t a b o l i t e s .

Hydroxylation sites of cannabinoids observed in vitro or in vivo

A d e t a i l e d d i s c u s s i o n o f many a l t e r n a t e methods f o r the q u a n t i t a t i v e d e t e r m i n a t i o n o f Δ -THC and some o f i t s m e t a b o l i t e s has been p r e s e n t e d p r e v i o u s l y ( 9 ) . T h i s p a p e r w i l l d e a l w i t h d e t e r m i n a t i o n o f A -THC, l i ­ ny droxy-Δ -THC and c a n n a b i n o l i n b l o o d w i t h one e x t r a c 9

9

9

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3.

W A L L

Detection and Quantitation

E T A L .

41

tion. I n a d d i t i o n , d e t a i l s w i l l be p r e s e n t e d f o r t h e d e t e r m i n a t i o n o f ll-nor-Δ -THC-9-carboxylic a c i d i n b l o o d and u r i n e . The l a t t e r m e t a b o l i t e i s g a i n i n g i n ­ creased importance i n f o r e n s i c d e t e r m i n a t i o n s . 9

9

9

METHODS FOR Δ -ΤΗΟ, CANNABINOL, AND 11-0H-A -THC Clinical

Protocol

Human, male v o l u n t e e r s who were e x p e r i e n c e d m a r i ­ j u a n a u s e r s were a d m i n i s t e r e d 4.0-5.0 mg o f A -THC by the i n t r a v e n o u s method o f Perez-Reyes e t a_l. (10) . The v o l u n t e e r s were k e p t under m e d i c a l s u p e r v i s i o n f o r 24 hours i n t h e C l i n i c a l Research U n i t o f t h e U n i v e r s i t y o f N o r t h C a r o l i n a , S c h o o l o f M e d i c i n e . B l o o d samples ( a p ­ p r o x i m a t e l y 10 ml) were c o l l e c t e d a t p e r i o d i c i n t e r v a l s o v e r 24 h o u r s . Plasma was o b t a i n e d by c e n t r i f u g a t i o n , f r o z e n i m m e d i a t e l y and s t o r e d i n f r o z e n c o n d i t i o n u n t i l analyzed.

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9

CH CH CH CH CR 2

Δ -THC; R - R 2

2

- H , R

2

3

1

R » ^3'

9

11- Η -Δ -ΤΗΟ; 3

9

A -THC-PFP, R - R

2

2

2

CH-CH.CH-CH-CR

2

ll-nor-Δ - T H C - 9 - c a r b o x y l i c a c i d ; R - H, 2 9 2 5 - Η -ll-nor-Δ - T H C - 9 - c a r b o x y l i c a c i d ; R - H, 2 8 ? 5'- H - l l - n o r - A - T H C - 9 - c a r b o x y l i c a c i d , R H

- Η R^ • H, R

2

- H

3

- H , Rj « O C C F ^ F j 3

3

9

ll-Hydroxy-A -THC; 2

ll-Hydroxy-5'- H

R - HjOH,

» H, R

9

-A -THC; R - H OH, R

£

« H

3

3

- H, R„

:H2CH2CH2CH2CH3

3a

C B N ; R - H, R j - H

b

5'- H3-CBN;

c

CBN-PFP;

2

R

R - H,

4a

3

R

J

m. O C C F 2 C F 3 ,

-

2

R

H J

b

3

Hexahydro-CBN;

R - Η

Hexahydro-CBN-PFP;

R -

OCCFJCHJ

-

Figure 2. Structure of cannabinoids and internal standards

Internal

Standards

A key f e a t u r e o f o u r q u a n t i t a t i v e p r o c e d u r e s was t h e use o f a p p r o p r i a t e d e u t e r a t e d a n a l o g s o f t h e canna­ b i n o i d s under s t u d y as b o t h c a r r i e r s f o r t h e s m a l l q u a n t i t y o f c a n n a b i n o i d s e x p e c t e d t o be p r e s e n t i n many c a s e s and as i n t e r n a l s t a n d a r d s f o r q u a n t i t a t i o n b y

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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CANNABINOID

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PHYSIOLOGICAL

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mass s p e c t r o m e t r y . The s t r u c t u r e s o f the c a n n a b i n o i d s and t h e i r d e u t e r a t e d analogs used i n t h e s e s t u d i e s are shown i n F i g u r e 2. A l l o f the compounds used were synt h e t i c and were made a v a i l a b l e by the N a t i o n a l I n s t i t u t e on Drug Abuse S y n t h e s i s Program.1 S y n t h e t i c methods f o r the v a r i o u s d e u t e r a t e d c a n n a b i n o i d s u t i l i z e d i n these s t u d i e s have been p r e s e n t e d by P i t t e t a l . (11).

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General Precautions C l o s e a t t e n t i o n must be p a i d t o the p r o c e d u r a l det a i l s p r e s e n t e d below i n o r d e r t o o b t a i n r e p r o d u c i b l e and q u a n t i t a t i v e d a t a . In g e n e r a l , i n working w i t h c a n n a b i n o i d s , exposure o f samples or e x t r a c t s t o l i g h t o r a i r s h o u l d be m i n i m i z e d . A l l solvent evaporations s h o u l d be conducted in_ vacuo o r under n i t r o g e n a t low temperature. Cannabinoids i n nanogram l e v e l s a r e subj e c t t o a d s o r p t i o n on the s u r f a c e of g l a s s w a r e . In o r d e r t o minimize t h i s problem a l l g l a s s w a r e , i n c l u d i n g chromatography columns, was s i l a n i z e d u s i n g 5% DMCS i n toluene. 9

A n a l y s i s o f A -THC, C a n n a b i n o l , and

9

11-0H-A -THC

E x t r a c t i o n and P u r i f i c a t i o n P r i o r t o A n a l y s i s by GLC-MS i n e l e c t r o n impact (EI) Mode — When the mass s p e c t r o m e t e r s were o p e r a t e d i n t h i s mode the m o l e c u l a r i o n s o r c h a r g e d fragments u t i l i z e d f o r the q u a n t i t a t i v e a n a l y s i s o f u n d e r i v a t i z e d c a n n a b i n o i d s were i n a range o f m/e 320 o r lower. P r e l i m i n a r y s t u d i e s w i t h plasma e x t r a c t s i n d i c a t e d t h a t i n t e r f e r e n c e from endogenous plasma c o n s t i t u e n t s would be e n c o u n t e r e d . This could be a v o i d e d by c a r r y i n g o u t a p r e l i m i n a r y c l e a n u p by Sephadex LH-20 chromatography p r i o r t o the GLC-MS s t e p . The methods which are p r e s e n t e d a r e f o r the combined d e t e r m i n a t i o n o f A -THC ( l a ) , l l - h y d r o x y - A - T H C (2a), and c a n n a b i n o l (3a) as shown i n F i g u r e 2. The methods, of c o u r s e , a r e e q u a l l y u t i l i z a b l e f o r the d e t e r m i n a t i o n of i n d i v i d u a l c o n s t i t u e n t s . Deuterated i n t e r n a l s t a n dards ( F i g u r e 2) were added t o a sample o f 3.0 ml o f c o l d (not f r o z e n ) plasma as f o l l o w s : l b , 150.0 ng; 2b, 15.0 ng; and 3b, 1.50 ng. Each i n t e r n a l s t a n d a r d 9

9

Research T r i a n g l e I n s t i t u t e C o n t r a c t HSM-42-71-95. Q u a l i f i e d i n v e s t i g a t o r s may o b t a i n l a b e l e d and u n l a b e l e d c a n n a b i n o i d s by a p p l i c a t i o n t o Dr. Robert W i l l e t t e , A c t i n g C h i e f , The Research Technology Branch, D i v i s i o n o f Research, NIDA, Rockwall B u i l d i n g , 11400 R o c k v i l l e P i k e , R o c k v i l l e , Maryland 20852.

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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ET

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was added i n 15-30 μΐ e t h a n o l . F o l l o w i n g a d d i t i o n o f each i n t e r n a l s t a n d a r d the plasma sample was s t i r r e d f o r 3-5 seconds i n a v o r t e x a g i t a t o r and then s u b j e c t e d t o s o n i c a t i o n (Cole-Parmer u l t r a s o n i c c l e a n e r ) f o r the same t i m e . The plasma samples ( c o n t a i n e d i n a screw capped c e n t r i f u g e tube) were t h e n e x t r a c t e d 3 t i m e s w i t h 6.0 ml p e t r o l e u m e t h e r (bp 30-60°, Nanogram Grade o r B u r d i c k and Jackson) c o n t a i n i n g 1.5% i s o a m y l a l c o ­ h o l . The t u b e s were a g i t a t e d 15 minutes each time i n a v o r t e x a g i t a t o r and the l a y e r s s e p a r a t e d by c e n t r i f u g a t i o n a f t e r each e x t r a c t i o n . The p e t r o l e u m e t h e r e x t r a c t s were combined, e v a p o r a t e d in_ vacuo a t room t e m p e r a t u r e and f r e e z e d r i e d o v e r n i g h t t o remove w a t e r and i s o a m y l a l c o h o l . The d r i e d r e s i d u e was d i s s o l v e d i n a m i n i m a l volume o f p e t r o l e u m e t h e r / c h l o r o f o r m / e t h a n o l (10:10:1) and chromatographed i n the same s o l v e n t m i x t u r e on 1 χ 40 cm w a t e r j a c k e t e d Sephadex LH-20 columns a t 26°C. Twenty-seven ml o f column e l u a n t were c o l l e c t e d and d i s c a r d e d . Seven ml o f e l u a n t was then c o l l e c t e d as t h e f r a c t i o n c o n t a i n i n g A -THC. The n e x t 8 ml o f e l u a n t was c o l l e c t e d as t h e C B N - c o n t a i n i n g f r a c t i o n . T h i r t y - e i g h t ml o f column e f f l u e n t was then c o l l e c t e d and d i s c a r d e d . F i n a l l y , 17 ml o f e l u a n t was c o l l e c t e d as t h e f r a c t i o n c o n t a i n i n g ll-hydroxy-Δ -THC. The A -THC and CBN f r a c t i o n s were e v a p o r a t e d t o d r y n e s s and d i s s o l v e d i n 30 μΐ hexane. The ll-hydroxy-Δ -THC f r a c t i o n was e v a p o r a t e d t o d r y n e s s under vacuum and h e a t e d w i t h 75 μΐ o f R e g i s i l (BSTFA + 1% TCMS) i n a c l o s e d v i a l a t 110° f o r 3 h o u r s . The r e a g e n t was r e ­ moved i n vacuum and t h e r e s i d u e d i s s o l v e d i n 20 μΐ hex­ ane. Gas Chromatography C o n d i t i o n s — On t h e LKB-9000 GLC-MS a 3 o r 6 χ 1/4" g l a s s column o f 2% OV-17 on Chromosorb W-HP (80/100 mesh) was u t i l i z e d , the former l e n g t h b e i n g used f o r ll-hydroxy-Δ -THC-bis-TMS e t h e r (at 220°C) and t h e l a t t e r l e n g t h f o r b o t h A -THC ( a t 220°C) and CBN ( a t 240°C). H e l i u m was used as the gas phase a t a r a t e of 35 ml/min. Under the c o n d i t i o n s s t i p u l a t e d above, r e t e n t i o n t i m e s o f 4-6 minutes were o b s e r v e d f o r each compound. Only Δ -THC and CBN were a n a l y z e d on t h e F i n n i g a n 3300 i n s t r u m e n t i n t h e E I mode. S i x f o o t g l a s s columns c o n t a i n i n g 1% SE-30 on 100/120 mesh Chromosorb W-HP were used a t column t e m p e r a t u r e s of 200-230°C and the f l o w o f 30-35 ml/min. Mass S p e c t r o m e t r y — The mass s p e c t r o m e t e r s and a s s o c i a t e d equipment have been d e s c r i b e d i n d e t a i l ( 9 ) . I n b r i e f , an LKB 9000 GLC-MS (a m a g n e t i c s e c t o r i n s t r u m e n t ) was u t i l i z e d w i t h a m o d i f i e d a c c e l e r a t i n g v o l t a g e a l t e r n a t o r (AVA) ( 1 2 ) . F o r Δ -THC the mass s p e c t r o m e t e r was s e t t o f o c u s a l t e r n a t e l y on the i o n s 9

9

9

9

f

f

9

9

9

9

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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m/e 314 and 317 w h i c h c o r r e s p o n d t o t h e m o l e c u l a r i o n o f t h e compound and i t s t r i d e u t e r o a n a l o g . F o r CBN the m o l e c u l a r i o n s were m/e 310 and 313. F o r a n a l y s i s o f ll-hydroxy-Δ -THC a s t h e bis-TMS e t h e r , t h e s t r o n g M-103 i o n (8, 13) a t m/e 371 a n d 374 was s e l e c t e d . The AVA a c c e s s o r y measures t h e two peak h e i g h t s o r a r e a s u t i l i z e d f o r each a n a l y s i s as d e s c r i b e d above. Alter­ n a t i v e l y t h e F i n n i g a n 3300 GLC/MS (a q u a d r u p o l e i n s t r u ­ ment) w i t h a d e d i c a t e d PDP-12 computer (9) was u t i l i z e d , and t h e r a t i o s o f peak h e i g h t s o r a r e a s were d e t e r m i n e d u s i n g computer s o f t w a r e . T L C - R a d i o - l a b e l P r o c e d u r e — The v o l u n t e e r s u b j e c t s ( d e s c r i b e d i n C l i n i c a l P r o t o c o l ) a l l r e c e i v e d 100 y C i o f t r i t i u m l a b e l e d A -THC, a l o n g w i t h t h e s t a n d a r d 4.05.0 mg i n t r a v e n o u s dose. Two t o t h r e e m l a l i q u o t s o f plasma were a n a l y z e d by t h e p r o c e d u r e d e s c r i b e d by W a l l (4) .

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9

9

Δ.Ο.Π-

8

io°

2

4

6

SAMPLES RUN ON DIFFERENT DAYS

Ίο

1

2

*

6

"ίο

2

NG NATURAL THC ADDED/50NG DEUTERATED THC

Finnigan 3300-EI plasma calibration curve for A -THC

Figure 3.

9

RESULTS FOR A -THC, CANNABINOL, AND l l - O H - A - T H C 9

Plasma c a l i b r a t i o n c u r v e s

9

o b t a i n e d w i t h t h e LKB

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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ET

Detection and Quantitation

AL.

45

and F i n n i g a n GLC-MS i n s t r u m e n t s were q u i t e s i m i l a r f o r b o t h A -THC and c a n n a b i n o l . F o r A -THC l i n e a r c a l i b r a ­ t i o n c u r v e s i n t h e range o f 1-100 ng/ml o f plasma were obtained w i t h both instruments. F i g u r e 3 shows d a t a f o r t h e F i n n i g a n . D e t e c t i o n o f A -THC down t o 0.1 n g / ml c o u l d be a t t a i n e d , b u t 0.5 ng/ml i s r e g a r d e d as t h e m i n i m a l c o n c e n t r a t i o n a t w h i c h r e l i a b l e d a t a c o u l d be obtained. Plasma c a l i b r a t i o n c u r v e s f o r CBN a r e exem­ p l i f i e d f o r t h e F i n n i g a n MS i n F i g u r e 4; t h e LKB d a t a was s i m i l a r . L i n e a r c u r v e s on b o t h i n s t r u m e n t s were o b t a i n e d between 0.2-10.0 ng/ml w i t h d e t e c t i o n l i m i t s a b o u t 0.1 ng/ml. Only the LKB 9 000 MS was used f o r t h e ll-hydroxy-Δ -THC determinations. The d a t a a r e shown i n F i g u r e 5. The c u r v e was l i n e a r i n t h e range o f 0.210.0 ng/ml plasma o f 11-hydroxy m e t a b o l i t e . 9

9

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9

9

10

1

I

Figure 4.

1

1—ι

r~|

1

1—i—r-|

1

1—r-τη

1

1—ι—τη

Finnigan 3300-EI plasma calibration curve for CBN

F i g u r e 6 p r e s e n t s t h e average v a l u e s w i t h s t a n d a r d e r r o r o b t a i n e d f o r A -THC, l l - O H - A - T H C and c a n n a b i n o l from plasma o f male v o l u n t e e r s r e c e i v i n g A -THC by i n ­ travenous i n f u s i o n . The measurements c o v e r e d a 24 h o u r p e r i o d . A -THC v a l u e s o b t a i n e d w i t h t h e LKB-9000 E I s o u r c e were i n c l o s e agreement w i t h t h e d a t a o b t a i n e d on the F i n n i g a n 3300 c h e m i c a l i o n i z a t i o n (CI) s o u r c e . 9

9

9

9

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Figure 5.

LKB 9000-EI plasma calibration curve for 11-hydroxy-A -THC 9

Comparison o f v a l u e s f o r LKB v s . F i n n i g a n EI s o u r c e a l s o showed e x c e l l e n t agreement. A -THC values i n c r e a s e d r a p i d l y d u r i n g the f i r s t 10-20 minutes, the peak v a l u e s i n t h e range o f 50-60 ng/ml c o i n c i d i n g w i t h the maximal psychomimetic a c t i v i t y . A typical biphasic e l i m i n a t i o n p a t t e r n was noted; the A -THC plasma l e v e l s d e c r e a s e d r a p i d l y between 15-40 minutes and t h e n f e l l a t a much s l o w e r r a t e . With a p a r t i c u l a r group o f v o l u n t e e r s (3 s u b j e c t s ) l e v e l s a f t e r 24 hours were between 3-5 ng/ml. Spot checks a t lower l e v e l s u t i l i z i n g the F i n n i g a n MID program c o n f i r m e d t h a t the substance b e i n g e v a l u a t e d was i n d e e d A - T H C and not i n s t r u m e n t "noise." In t h e case o f l l - h y d r o x y - A - T H C much lower l e v e l s were found. Peak v a l u e s i n the n e i g h b o r h o o d of 2.0 ng/ml were n o t e d between 30-40 m i n u t e s . The maximal v a l u e s d e c l i n e d i n a more g r a d u a l manner than was 9

9

9

9

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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3.

W A L L

E T

A L .

Detection and Quantitation

o.o - ο

I

0

47

0

1

1

I

I

I

I

I

10

0

10

20

30

40

50

I

60

I

1

1

70

80

90

, ,

Lh 1440

MINIITFS

Figure 6. Plasma levels of A -THC, ll-hydroxy-A -THC, and CBN found over a 24-hr period in human plasma from volunteers receiving A -THC by iv adminis­ tration 9

9

9

9

the case f o r A -THC, f a l l i n g t o a l e v e l o f 1.0 ng/ml i n 60-90 minutes and 0.5 ng/ml a f t e r 24 h o u r s . The v a l u e s f o r CBN shown i n F i g u r e 6 have no p h a r m a c o k i n e t i c s i g ­ n i f i c a n c e a s most o f t h e d a t a f a l l s below t h e l e v e l o f analytical reliability. F i g u r e 7 compares t h e r e s u l t s o b t a i n e d from t h e average o f f o u r s u b j e c t s a n a l y z e d by GLC-MS-EI, TLC, r a d i o - l a b e l , and e l e c t r o n c a p t u r e GLC. C o r r e l a t i o n c o ­ e f f i c i e n t s a r e c a l c u l a t e d i n F i g u r e 8. The r e s u l t s a r e i n r e a s o n a b l e agreement, and i n p a r t i c u l a r t h e GLC-MS and e l e c t r o n c a p t u r e GLC p r o c e d u r e s gave good agreement f o r most p o i n t s over t h e whole c u r v e . 9

METHODS FOR ll-NOR-Δ -THC-9-CARBOXYLIC ACID E x t r a c t i o n and P u r i f i c a t i o n F o l l o w i n g t h e a d d i t i o n o f 300 ng o f d e u t e r a t e d i n t e r n a l s t a n d a r d (5c, c f . F i g . 2) t o 3.0 ml o f c o l d plasma o r u r i n e , t h e sample was s o n i c a t e d w i t h 30 m l

American Chemical Society Library '1155 16th St. N. W. Washington, D. C. 20036

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

48

CANNABINOID

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80

ANALYSIS

IN

PHYSIOLOGICAL

FLUIDS

f-

MINUTES

Figure 7.

The A -THC found in plasma following administration of 5 mg A -THC (average of 4 subjects) 9

9

a c e t o n e f o r 30 m i n u t e s . The sample was c e n t r i f u g e d t o remove p r e c i p i t a t e d p r o t e i n s , t h e a c e t o n e d e c a n t e d , and t h e p e l l e t r e - e x t r a c t e d w i t h an a d d i t i o n a l 30 ml a c e t o n e . Acetone e x t r a c t s were combined and c o n c e n t r a t e d in_ vacuo t o an aqueous r e s i d u e . The volume was a d j u s t e d t o 3.0 ml w i t h d i s t i l l e d w a t e r and t h e pH t o 3. The c a n n a b i n o i d a c i d s were e x t r a c t e d from t h e aqueous phase by two p a r t i t i o n s w i t h 10 ml o f d i e t h y l e t h e r . T h i s was a c c o m p l i s h e d by a g i t a t i n g t h e c e n t r i fuge t u b e s 15 m i n u t e s each t i m e i n a v o r t e x a g i t a t o r and s e p a r a t i n g t h e phases by c e n t r i f u g a t i o n . The d i e t h y l e t h e r e x t r a c t s were combined, e v a p o r a t e d i n vacuo, r e - d i s s o l v e d i n 0.5 ml m e t h a n o l , and f i l t e r e d through a 5 micron t e f l o n f i l t e r .

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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3.

W A L L E T AL.

Detection and Quantitation

49 METHODS

CORRELATION

COMPARED

COEFFICIENT

NG Λ -THC/ML PLASMA

Figure 8. Least-squares best lines comparing all data obtained by each of two methods of analysis

The e x t r a c t s thus o b t a i n e d were p u r i f i e d by h i g h p e r f o r m a n c e l i q u i d chromatography (HPLC) on a r e v e r s e phase y - C ( P a r t i s i l - 2 0 ) column (30 cm χ 4.6 mm ID) u s i n g a m e t h a n o l / w a t e r m o b i l e phase (65:35, c o n t a i n i n g 0.05% ammonium a c e t a t e ) . A t a f l o w r a t e o f 4 ml/min. the m i x t u r e o f 5a and d e u t e r a t e d i n t e r n a l s t a n d a r d 5c were e l u t e d a t a time i n t e r v a l o f 8-13 m i n u t e s . This f r a c t i o n o f e l u a n t was c o l l e c t e d and f r e e z e d r i e d . The r e s i d u e was t r a n s f e r r e d t o a h a l f dram v i a l and d r i e d i n vacuo o v e r n i g h t w i t h 10 mg o f p o t a s s i u m c a r b o n a t e . Under anhydrous c o n d i t i o n s 0.2 m l d r y a c e t o n e and 8 y l d i m e t h y l s u l f a t e were added t o the sample. The v i a l was t i g h t l y capped and h e a t e d w i t h s h a k i n g a t 50°C f o r 6 hours. F o l l o w i n g the a d d i t i o n o f 0.5 m l d i s t i l l e d w a t e r t o e a c h sample, the d e r i v a t i z e d c a n n a b i n o i d s were removed by two e x t r a c t i o n s w i t h 0.5 m l c h l o r o f o r m . The c h l o r o f o r m was e v a p o r a t e d under n i t r o g e n and the r e s i ­ due r e - d i s s o l v e d i n 30 y l c h l o r o f o r m . 1 8

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

50

CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS

GLOMS A n a l y s i s The d i m e t h y l a t e d d e r i v a t i v e s o f t h e m i x t u r e o f 5a and 5c were s u b m i t t e d t o a n a l y s i s on t h e LKB-9000 G L O MS w i t h AVA d e s c r i b e d p r e v i o u s l y . Samples were chromat o g r a p h e d a t 250°C on a 6 column o f OV-17, 2%, on S u p e l c o p o r t (80/100 mesh) w i t h a He c a r r i e r gas f l o w r a t e o f 30 ml/minute. The r a t i o o f t h e peak h e i g h t s a t m/e 3 72 and 375 was d e t e r m i n e d i n t h e u s u a l manner. Downloaded by UNIV OF CALIFORNIA SAN DIEGO on July 14, 2016 | http://pubs.acs.org Publication Date: April 10, 1979 | doi: 10.1021/bk-1979-0098.ch003

1

0.5

\-

O

I

1 I I I I

I

1

I

I I I

I

10°

I I I I I 1 10

I

I

I

I

•• • •I » 10

1

2

NG 11 NOR A - T H C 9 COOH/ML 9

Figure 9.

Instrument calibration for ll-nor-A -THC-9-COOH human plasma 9

extracted from

NG D / M L 0

Figure 10.

Instrument calibration curve for ll-nor-A -THC-9-COOH urine 9

in human

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3.

WALL ET AL.

Detection and Quantitation

51

9

RESULTS FOR ll-NOR-Δ -THC-9-CARBOXYLIC ACID A plasma c a l i b r a t i o n c u r v e f o r l l - n o r - A - T H C - 9 c a r b o x y l i c a c i d , 5 a , i s shown i n F i g u r e 9. There was r e a s o n a b l e l i n e a r i t y from 1.0-50 ng/ml plasma w i t h de­ t e c t i o n l i m i t s o f 0.5 ng o r l e s s p e r m l . F i g u r e 10 p r e ­ sents s i m i l a r data f o r a u r i n e c a l i b r a t i o n curve. The method showed r e a s o n a b l e l i n e a r i t y between 2.0-100 n g / ml u r i n e . F i g u r e 11 p r e s e n t s p h a r m a c o k i n e t i c d a t a f o r plasma l e v e l s o f a human v o l u n t e e r , BS, o v e r a 0.5 h o u r t o 48 hour p e r i o d comparing A -THC and 11-nor a c i d l e v e l s a f t e r a dose o f 5.0 mg o f A -THC by t h e i n t r a ­ venous r o u t e . B o t h p a r e n t compound and a c i d m e t a b o l i t e e x h i b i t e d a biphasic e l i m i n a t i o n pattern although the l e v e l s o f t h e a c i d d i d n o t f a l l as r a p i d l y as p a r e n t compound. E l i m i n a t i o n o f t h e a c i d m e t a b o l i t e 5a i n u r i n e i s shown i n F i g u r e 12. I t i s e v i d e n t t h a t u r i n a r y e l i m i n a t i o n p r o c e e d e d r a p i d l y as 80% o f t h e t o t a l 11n o r - a c i d e x c r e t e d was e l i m i n a t e d i n t h e u r i n e d u r i n g 9

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9

9

Δ 11

.

I 10

ι

I

,

20

I

ι

I

30

ι

40

HOURS

Figure 11.

The A -THC and ll-nor-A -THC-9-COOH excreted in the urine of subject BS after iv infusion of 4 mg A -THC 9

9

9

the f i r s t 6 hours a f t e r a d m i n i s t r a t i o n o f p a r e n t com­ pound; f u r t h e r e l i m i n a t i o n p r o c e e d e d much more s l o w l y . T a b l e 1 compares r e s u l t s o b t a i n e d by TLC a s s a y (4) and the GLC-MS p r o c e d u r e i n u r i n e and plasma o f s u b j e c t BS. Agreement was e x c e l l e n t i n most o f t h e c a s e s .

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS

HOURS

Figure 12.

The ll-nor-A -THC-9-COOH excreted in the urine of subject BS after iv infusion of 4 mg A -THC 9

9

Table 1 Comparison of GLC-MS and TLC Analysis of 11-nork -THC-9-C00H in the Plasma and Urine of Subject BS . 9

ng/ml p l a s m a

f o u n d by

minutes glc-ms

tic

30

36

36

60

30

27

90

32

28

120

35

25

ng/ml

urine

f o u n d by

hours tic

3 6

7.4 15

glc-ms

5.4 22

12

6.7

6.2

24

2.1

.2

48

1.0

72

0.89

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3. W A L L E T AL.

Detection and Quantitation

53

DISCUSSION The b a s i c o b j e c t i v e o f t h i s i n v e s t i g a t i o n was t o e s t a b l i s h s e n s i t i v e methodology w h i c h w o u l d n o t depend on r a d i o - l a b e l i n g f o r t h e q u a n t i t a t i v e e s t i m a t i o n o f A -THC, i t s p r i m a r y m e t a b o l i t e ll-hydroxy-A -THC (3), and c a n n a b i n o l , w h i c h has been r e p o r t e d t o be a metabol i t e o f A -THC i n t h e r a t ( 1 4 ) . T h i s o b j e c t i v e has been r e a l i z e d , u t i l i z i n g GLC-MS w i t h a v a r i e t y o f t e c h n i q u e s and i n s t r u m e n t s . In a d d i t i o n , the q u a n t i t a t i v e estimat i o n o f l l - n o r - A - T H C - 9 - c a r b o x y l i c a c i d has been accomp l i s h e d . Several aspects of our r e s u l t s merit f u r t h e r discussion. 9

9

9

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9

Choice o f Instrument Two c o m p l e t e l y d i f f e r e n t t y p e s o f mass s p e c t r o m e t e r s c o u p l e d w i t h d i f f e r e n t means f o r q u a n t i t a t i o n o f d a t a were u t i l i z e d . One was a r e l a t i v e l y o l d (1968) m a g n e t i c s e c t o r MS, t h e LKB-9000, w h i c h was c o u p l e d w i t h an a c c e l e r a t i n g v o l t a g e a l t e r n a t o r w h i c h p e r m i t t e d measurement o f t h e r a t i o o f t h e peak h e i g h t o f t h e unknown as compared w i t h t h a t o f t h e i n t e r n a l s t a n d a r d . The o t h e r was a newer (19 74) q u a d r u p o l e MS, t h e F i n n i g a n 3300, w h i c h was i n t e r f a c e d t o a PDP-12 computer. The F i n n i g a n MS has b o t h E I and CI s o u r c e s . As shown i n t h e R e s u l t s s e c t i o n , b o t h i n s t r u m e n t s i n t h e E I mode gave v i r t u a l l y i d e n t i c a l plasma c a l i b r a t i o n c u r v e s w i t h i d e n t i c a l l i n e a r range and q u i t e s i m i l a r s t a n d a r d e r r o r o f e s t i m a t i o n . F i g u r e 6 g i v e s p h a r m a c o k i n e t i c d a t a i n man o b t a i n e d on t h e LKB i n t h e E I mode and t h e F i n n i g a n i n the C I mode. The r e s u l t s a r e q u i t e s i m i l a r . I t i s thus e v i d e n t t h a t a w i d e v a r i e t y o f mass s p e c t r o m e t e r s can be used w i t h comparable r e s u l t s p r o v i d e d a p p r o p r i a t e i n t e r n a l c a r r i e r s and s t a n d a r d s a r e added. B e f o r e c o n c l u d i n g t h i s d i s c u s s i o n one word o f c a u t i o n s h o u l d be g i v e n . The n a t u r e o f t h e s e p a r a t o r s i s most i m p o r t a n t ; t h e LKB w i t h t h e Ryhage s e p a r a t o r and t h e F i n n i g a n w i t h a s i l y l a t e d g l a s s j e t s e p a r a t o r gave a p p r o p r i a t e s e n s i t i v i t y . On t h e o t h e r hand, a n o t h e r mass s p e c t r o m e t e r w h i c h u t i l i z e d a Watson-Biemann s e p a r a t o r showed p o o r s e n s i t i v i t y and c o u l d n o t be u t i l i z e d f o r c a n n a b i n o i d s t u d i e s . I n t e r n a l Standards As i n d i c a t e d p r e v i o u s l y , t h e f i n a l mass s p e c t r o m e t r i c measurements c a n be c o n d u c t e d w i t h g r e a t a c c u r a c y . The k e y t o s u c c e s s i n t h e v a r i o u s a n a l y t i c a l s t u d i e s was t h e u t i l i z a t i o n o f a p p r o p r i a t e compounds w h i c h c o u l d be employed as b o t h c a r r i e r s and i n t e r n a l s t a n d a r d s .

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

54

CANNABINOID

ANALYSIS

IN PHYSIOLOGICAL

FLUIDS

F o r t h i s purpose deuterium l a b e l e d c a n n a b i n o i d s i d e n t i c a l t o the p a r e n t compound except f o r the l a b e l a r e i d e a l and were u t i l i z e d f o r a l l o f t h e E I s t u d i e s . I t i s p o s s i b l e t o use w i t h e q u a l s u c c e s s an i n t e r n a l c a r r i e r which i s n o t i s o t o p i c a l l y l a b e l e d as long as i t s p r o p e r t i e s a r e very s i m i l a r t o t h a t o f the cannabinoid b e i n g s t u d i e d b u t p e r m i t s e p a r a t i o n by GLC. Hexahydroc a n n a b i n o l was e x c e l l e n t f o r t h i s purpose and was used i n CI s t u d i e s o f A -THC and c a n n a b i n o l . 9

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The

A n a l y s i s f o r l l - N o r - A - T H C - 9 - C a r b o x y l i c A c i d (5a) 9

The a n a l y s i s f o r t h i s "end" m e t a b o l i t e o f A 9 _ caused p a r t i c u l a r problems. E s p e c i a l l y b a f f l i n g f o r a c o n s i d e r a b l e time was the a n a l y s i s o f 5a i n plasma. T h i s was f i n a l l y s o l v e d by the r e a l i z a t i o n t h a t 5a. i n plasma o r u r i n e c o n t a i n i n g b l o o d ( i n cases o f i l l n e s s o r i n j u r y ) i s p r o b a b l y n o n - c o v a l e n t l y bonded t o a p r o tein. As a r e s u l t , a l t h o u g h t h i s combination i s e t h e r e x t r a c t a b l e , i t remains bound and passes through t h e r e q u i s i t e HPLC p u r i f i c a t i o n s t e p a t a r e t e n t i o n time mark e d l y d i f f e r e n t than pure 5^. I t was found t h a t t r e a t ment o f t h e " p r o t e i n " - 5 a complex w i t h a c e t o n e p r e c i p i t a t e d t h e plasma p r o t e i n s and broke up t h e "protein"-5a. complex. Other p o i n t s o f i n t e r e s t were t h e requirement f o r d e r i v a t i z a t i o n o f 5a as t h e m e t h y l e s t e r o f t h e c a r b o x y l i c a c i d and t h e methyl e t h e r o f t h e p h e n o l i c hydroxy 1 m o i e t y . B i s - s i l y l a t i o n o f these f u n c t i o n s was n o t q u a n t i t a t i v e . The A - d e u t e r a t e d a n a l o g 5a i s more r e a d i l y a v a i l a b l e than the A - d e u t e r a t e d a n a l o g 5b. R e c e n t l y the l a t t e r has been s y n t h e s i z e d (11, 1 6 ) . No advantage i n p r e c i s i o n o r s e n s i t i v i t y was n o t e d i n u s i n g 5b i n p r e f e r e n c e t o 5 c . T H C

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M e t a b o l i c and P h a r m a c o k i n e t i c

Data

The development o f s e n s i t i v e and a c c u r a t e GLC-MS methodology p e r m i t t e d a p r e l i m i n a r y study i n man u t i l i z i n g these t e c h n i q u e s f o r the p r e c i s e d e t e r m i n a t i o n o f A -THC, 11-hydroxy-A -THC and CBN i n plasma. P r e v i o u s l y we have made an e x t e n s i v e study (4) o f t h e metabolism o f A -THC i n man u s i n g r a d i o - l a b e l e d t r a c e r s and t h i n l a y e r chromatography. The p r o c e d u r e s u t i l i z e d ( i n a d d i t i o n t o the u n d e s i r a b i l i t y o f a r a d i o - l a b e l e d t r a c e r i n man) s u f f e r from two p o t e n t i a l s o u r c e s o f e r r o r . The method would n o t p e r m i t s e p a r a t i o n o f A -THC from CBN, and i n t h e case o f 11-hydroxy- A9-THC, would n o t p e r m i t s e p a r a t i o n from o t h e r monohydroxy m e t a b o l i t e s which might be p r e s e n t (8, 1 7 ) . The data i n F i g u r e 6 f o r A -THC a r e q u i t e comparable 9

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to pharmacokinetic data o b t a i n e d i n e a r l i e r s t u d i e s (4). In both instances a b i p h a s i c e l i m i n a t i o n curve was n o t e d . A f t e r the i n i t i a l maximum l e v e l , a s h a r p d e c l i n e was f o l l o w e d by a more g r a d u a l d e c r e a s e . Maxi­ mal v a l u e s i n the c u r r e n t s t u d i e s were 50-60 ng/ml. A f t e r 24 h o u r s , 3-5 ng/ml o f A - T H C were s t i l l found i n the plasma. Our r e s u l t s f o r 11-hydroxy-Δ9-THC a r e p r o ­ b a b l y the most a c c u r a t e d a t a y e t r e p o r t e d i n man. The c o n c e n t r a t i o n o f t h i s a c t i v e m e t a b o l i t e ( F i g u r e 6) was o n l y 2-3 ng/ml a t peak l e v e l s d e c l i n i n g a t a s l o w e r r a t e t h a n Δ -THC t o 0.5 ng/ml a f t e r 24 h o u r s . A l t h o u g h Δ THC i s r e a d i l y c o n v e r t e d t o l l - h y d r o x y - Δ - T H C i n t h e l i v e r ( 3 ) , o n l y s m a l l q u a n t i t i e s f i n d t h e i r way i n t o the b l o o d . Our i n t e r e s t i n CBN was a r o u s e d by r e p o r t s from McCallum (14, 15) w h i c h i n d i c a t e t h a t CBN m i g h t be a t r a n s i t o r y m e t a b o l i t e found a t v e r y e a r l y time p e r i o d s a f t e r a d m i n i s t r a t i o n o f A 9 - T H C . AS shown i n F i g u r e 6, the l e v e l o f CBN was below the r e l i a b i l i t y l i m i t s i n the E I mode. Other s t u d i e s we have c a r r i e d o u t by e l e c t r o n c a p t u r e GLC o r GLC-MS i n t h e CI mode i n d i c a t e the v i r t u a l absence o f t h i s s u b s t a n c e a t a l l time p e r i ­ ods. S i n c e we have found t h a t CBN has the same g e n e r a l p h a r m a c o k i n e t i c p a t t e r n as Δ - Τ Η 0 i n man ( 4 ) , we must c o n c l u d e t h a t CBN can be d i s r e g a r d e d i n terms o f i t s i m p o r t a n c e as a m e t a b o l i t e i n man. We p r e s e n t f o r t h e f i r s t t i m e p h a r m a c o k i n e t i c d a t a i n plasma and u r i n e o b t a i n e d by GLC-MS f o r the i m p o r t a n t acid metabolite ll-nor-Δ -THC-9-carboxylic acid. This and r e l a t e d a c i d s c o n s t i t u t e t h e major means by w h i c h A - T H C i s e x c r e t e d i n the u r i n e . The d a t a i n d i c a t e r a p i d e l i m i n a t i o n o f t h e a c i d i n the u r i n e d u r i n g t h e f i r s t 3-6 hours a f t e r a d m i n i s t r a t i o n o f A - T H C . 9

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Comparison o f GLC-MS W i t h Other

Procedures

As shown i n F i g u r e s 7 and 8, t h e GLC-MS p r o c e d u r e s show r e a s o n a b l e agreement i n the case o f Δ -THC w i t h d a t a o b t a i n e d by two i n d e p e n d e n t p r o c e d u r e s i n v o l v i n g , r e s p e c t i v e l y , t h i n l a y e r chromatography o f r a d i o - l a b e l e d c a n n a b i n o i d s and a double G L C - e l e c t r o n c a p t u r e p r o c e ­ dure. I n p r e l i m i n a r y s t u d i e s on c a n n a b i n o l l e v e l s o f s u b j e c t s who r e c e i v e d A9-THC, good agreement was found between the GLC-MS E I method, r e i n f o r c i n g our b e l i e f t h a t CBN i s n o t a s i g n i f i c a n t m e t a b o l i t e o f Δ -THC i n man. F i n a l l y , T a b l e 1 shows e x c e l l e n t agreement be­ tween t h e T L C - r a d i o - l a b e l e d p r o c e d u r e and GLC-MS a n a l y ­ s i s f o r t h e 1 1 - n o r - a c i d , 5a. 9

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ACKNOWLEDGMENTS These studies were conducted with the support of the N a t i o n a l I n s t i t u t e on Drug Abuse under contracts HMS-41-75-95 and ADM-45-74-109. We wish to thank Mario Perez-Reyes, M.D. for c l i n i c a l m a t e r i a l used i n some of these studies and express to Mrs. V a l e r i e H. S c h i n d l e r , Mr. P h i l i p Brown B e l t and Mr. Jarvey M. Tay­ l o r our a p p r e c i a t i o n for t h e i r t e c h n i c a l a s s i s t a n c e .

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REFERENCES (1) (2) (3) (4) (5)

(6) (7)

(8)

(9)

(10) (11) (12) (13)

Mechoulam, R . , Ed., "Marijuana", Academic P r e s s , New York, New York, 1973, pp. 1-409. Paton, W. D. and Crown, J., "Cannabis and I t s De rivatives", Oxford U n i v e r s i t y Press, London, England, 1972, pp. 1-198. W a l l , M. E . i n "Recent Advances i n Phytochemis t r y " , V . C. Runeckles, E d . , Plenum P u b l i s h i n g C o . , New York, 1975, pp. 29-61. W a l l , M. E., B r i n e , D. R. and Perez-Reyes, M., in "Pharmacology of Marijuana", Eds. M. Braude and S. Szara, Raven P r e s s , New York, 1976, pp. 93-116. Lemberger, L., i n "Advances i n Pharmacology and Chemotherapy", Eds. G a r r a t i n i , S., F . Hawking, A. Golden, and I. Kopin, Academic Press, New York, 1972, p . 221. Hammar, C . - G . and Holmstedt, B . , Anal. Biochem. 22, 532 (1968). A g u r e l l , S . , Gustafsson, B . , Holmstedt, B., Lean der, K . , Lindgren, J-E., N i l s s o n , I., Sandberg, F. and Asberg, M., J. Pharm. Pharmac. 25, 554 (1973). W a l l , M. E., B r i n e , D. R. and Perez-Reyes, M., in "Marijuana: Chemistry, Biochemistry, and Cellular E f f e c t s " , Ed. G. G. Nahas, SpringerV e r l a g , New York, 1976, pp. 51-62. W a l l , M. E., Harvey, T. M . , Bursey, J. T . , B r i n e , D. R. and Rosenthal, D . , i n "Cannabinoid Assays in Humans", NIDA Research Monograph No. 7, 1976, pp. 107-117. Perez, Reyes, M . , Timmons, M., L i p t o n , M . , Davis, K. and W a l l , M . , Science 177, 633 (1972). Pitt, C. G . , Hobbs, D. T . , Schran, H . , Twine, C. E . and W i l l i a m s , D. L., J. Label Comp. 11, 551 (1975). K l e i n , P . D . , Hauman, J. R. and Εisler, W. J., Anal. Chem. 44, 410 (1972). W a l l , M. E., B r i n e , D. R . , B r i n e , G. Α . , Pitt, C. G . , Freudenthal, R. I . and Christensen, H. D . ,

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3. W A L L E T AL.

Detection and Quantitation

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J. Amer. Chem. Soc. 92, 3466 (1970). (14) McCallum, Ν. K., Experientia 31, 957 (1975). (15) McCallum, Ν. Κ., Yagen, Β . , Levy, S. and Mechou lam, R . , Experientia 31, 510 (1975). (16) Pitt, C. G., Fowler, M . , Sathe, S . , S r i v a s t a v a , S. C. and W i l l i a m s , D. L., J. Amer. Chem. Soc. 97, 3798 (1975). (17) W a l l , Μ. Ε. and B r i n e , D. R . , "Summaries", I n t e r n a t i o n a l Symposium on Mass Spectrometry in Biochemistry and Medicine, M i l a n , Italy, 1973, p . 52. RECEIVED

December

12, 1978.

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.